EPR study of ferric native prostaglandin H synthase and its ferrous NO derivative

Purified prostaglandin H synthase (EC 1.14.99.1) apoprotein, a polypeptide of 72 kDA, was titrated with hemin and EPR spectra of high-spin ferric heme were observed at liquid-helium temperature. With up to one hemin per polypeptide, a signal at g = 6.6 and 5.4, rhombicity 7.5%, evolved owing to specifically bound, catalytic active heme. At higher heme/polypeptide ratios signals at g = 6.3 and 5.9 were observed which were assigned to non-specific heme with no catalytic function. In microsomes from ram seminal vesicles the native enzyme showed the signal at g = 6.7 and 5.2 which could not be increased by the addition of hemin. Cyanide, an inhibitor of the enzyme, reacted at lower concentrations with the specific heme abolishing its signal at g = 6.6 and 5.4. Higher concentrations of cyanide were needed for the disappearance of the signal of non-specific heme. The reduced enzyme reacted with NO and formed two types of NO complexes. A transient complex, with a rhombic signal at gx = 2.07, gz = 2.01 and gy = 1.97, was assigned to a six-coordinate complex. The final, stable complex showed an axial signal at g = 2.12 and g = 2.001 and was assigned to a five-coordinate complex, where the protein ligand was no longer bound to the heme iron. Neither type of signal showed a hyperfine splitting from nitrogen of histidine indicating the absence of a histidine-iron bond in the enzyme. From these results and the similarity of the EPR signal at g = 6.6 and 5.4 to the signal of native catalase (EC 1.11.1.6) we speculated that tyrosinate might be the endogenous ligand of the heme in prostaglandin H synthase.     

Chemical modification of prostaglandin endoperoxide synthase by N-acetylimidazole. Effect on enzymic activities and EPR spectroscopic properties.

Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by N-acetylimidazole under conditions typical for the O-acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22 degrees C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N-acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 +/- 0.4 for the apoprotein and 1.0 +/- 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g = 6.7 and 5.3) when hemin was added to the N-acetylimidazole-reacted apoprotein. Treatment of N-acetylimidazole-reacted apoprotein with hydroxylamine restored the specific binding of heme. The N-acetylimidazole-reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical. These results demonstrate that heme protects against the inactivating modification by N-acetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effects. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N-acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D. L. and Smith, W. L. (1988) Proc. Natl Acad. Sci. USA 85, 1412-1416 including a signal peptide of 24 residues which is missing in the processed protein.     

Heme prosthetic group required for acetylation of prostaglandin H synthase by aspirin

The ability of aspirin to acetylate PGH synthase was determined by reacting [3H-acetyl]-aspirin with purified enzyme followed by high pressure liquid chromatography analysis of the protein components of the reaction mixture. Heme-reconstituted enzyme incorporated approximately one acetyl group per 70-kDa subunit, whereas apoprotein incorporated 0.1 acetyl group per subunit. The ability of the heme prosthetic group to enhance acetylation of the protein was correlated with its ability to protect the Arg253-Gly254 peptide bond from cleavage by trypsin. Thus, heme-induced alteration of protein conformation may contribute to the enhanced labeling of Ser506 by aspirin. The present results indicate that irreversible inactivation of prostaglandin H synthase by aspirin occurs only when the heme prosthetic group is bound to the protein. Considering its short in vivo half-life, it is likely that aspirin inactivates only the steady-state fraction of PGH synthase in a cell that is active but not newly synthesized apoprotein. This may contribute to the differential kinetics of inactivation and recovery of PGH synthase activity in platelets and vascular endothelial cells after administration of low dose aspirin as a prophylactic agent against cardiovascular disease.     

EPR spectroscopic characterization of an 'iron only' nitrogenase. S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus.

The alternative nitrogenase of Rhodobacter capsulatus, isolated from a nifHDK deletion mutant, has been purified to near homogeneity and identified as an 'iron only' nitrogenase. The dithionite-reduced component 1 ('FeFe protein') of this enzyme showed an EPR spectrum consisting of two components: a minor S = 1/2 signal at g = 1.93 and a very characteristic S = 3/2 signal of near-stoichiometric intensity at g = 5.44. This resonance is very close to the highest possible g value (g = 5.46) for the coinciding two intradoublet subspectra of an S = 3/2 system of maximal rhombicity (E/D = 0.33). The deviation from axial symmetry (increasing E/D) correlates with the stability, activity and substrate selectivity of the different (Mo, V, Fe) nitrogenases.     

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex.

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Cytochrome c-554 from Methylosinus trichosporium OB3b; a protein that belongs to the cytochrome c2 family and exhibits a HALS-Type EPR signal

A small soluble cytochrome c-554 purified from Methylosinus trichosporium OB3b has been purified and analyzed by amino acid sequencing, mass spectrometry, visible, CD and EPR spectroscopies. It is found to be a mono heme protein with a characteristic cytochrome c fold, thus fitting into the class of cytochrome c(2), which is the bacterial homologue of mitochondrial cytochrome c. The heme iron has a Histidine/Methionine axial ligation and exhibits a highly anisotropic/axial low spin (HALS) EPR signal, with a g(max) at 3.40, and ligand field parameters V/ξ = 0.99, Δ/ξ = 4.57. This gives the rhombicity V/Δ = 0.22. The structural basis for this HALS EPR signal in Histidine/Methionine ligated hemes is not resolved. The ligand field parameters observed for cytochrome c-554 fits the observed pattern for other cytochromes with similar ligation and EPR behaviour.     

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.     

The interaction of myeloperoxidase with ligands as studied by EPR

1. The reaction of myeloperoxidase with fluoride, chloride and azide has been studied by EPR. 2. Fluoride decreases the rhombicity of the high-spin heme signal of myeloperoxidase and the nuclear spin of the fluoride atom induces a splitting in g parallel of 35 G. This observation demonstrates that fluoride binds as an axial ligand to the heme iron of the enzyme. 3. Addition of chloride to the fluoride-treated enzyme increases the rhombicity of the high-spin heme signal and brings about a disappearance of the splitting at g parallel. The addition of azide to the fluoride-treated enzyme changes the spin state of the heme iron from a high-to a low-spin state (gx = 2.68, gy = 2.22 and gz = 1.80). 4. Upon addition of chloride or fluoride to low-spin azido-myeloperoxidase this compound is converted into the high-spin chlorido- or fluorido-myeloperoxidase. These observations demonstrate that these ligands compete for a binding site at or close to the heme iron of myeloperoxidase.     

Functional differentiation of cyclooxygenase and peroxidase activities of prostaglandin synthase by trypsin treatment. Possible location of a prosthetic heme binding site

Treatment of prostaglandin (PG)H synthase purified from ram seminal vesicle microsomes with trypsin cleaves the 70-kDa subunits into 33- and 38-kDa fragments (Chen, Y.-N. P., Bienkowski, M. J., and Marnett, L. J. (1987) J. Biol. Chem. 262, 16892-16899). In contrast to a minimal decrease in cyclooxygenase activity, peroxidase activity declines rapidly following trypsin treatment. The time course for loss of guaiacol peroxidase activity corresponds closely to the time course for protein cleavage. The ability of trypsin-treated enzyme to support catalytic reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of reducing substrates is significantly reduced. The products of metabolism of 10-hydroperoxy-8,12-octadecadienoic acid indicate that trypsin-treated enzyme catalyzes homolytic scission of the hydroperoxide bond in contrast to the heterolytic scission catalyzed by intact enzyme. Spectrophotometric titrations of hematin addition to trypsin-treated PGH synthase indicate approximately a 50% reduction in heme binding. These observations suggest that trypsin treatment of PGH synthase decreases the ability of the protein to bind prosthetic heme at a site that controls peroxidase activity. Comparison of the N-terminal sequence of the 38-kDa fragment of trypsin-treated PGH synthase to the amino acid sequence of the intact protein indicates that cleavage occurs between Arg253 and Gly254. Based on literature precedents and the results of the present investigations, we propose that the heme prosthetic group that controls the peroxidase activity of PGH synthase binds to the His residue of the sequence His250-Tyr251-Pro252-Arg253 located immediately adjacent to the trypsin cleavage site.     

Heme spin states and peroxide-induced radical species in prostaglandin H synthase

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.     

EPR characterization of a high-spin system in carbon monoxide dehydrogenase from Methanothrix soehngenii.

Carbon monoxide dehydrogenase and methyl-coenzyme M reductase were purified from 61Ni-enriched and natural-abundance nickel-grown cells of the methanogenic archae Methanothrix soehngenii. The nickel-EPR signal from cofactor F-430 in methyl-CoM reductase was of substoichiometric intensity and exhibited near-axial symmetry with g = 2.153, 2.221 and resolved porphinoid nitrogen superhyperfine splittings of approximately 1 mT. In the spectrum from 61Ni-enriched enzyme a well-resolved parallel I = 3/2 nickel hyperfine splitting was observed, A parallel = 4.4 mT. From a computer simulation of this spectrum the final enrichment in 61Ni was estimated to be 69%, while the original enrichment of the nickel metal was 87%. Carbon monoxide dehydrogenase isolated from the same batch exhibited four different EPR spectra. However, in none of these signals could any splitting or broadening from 61Ni be detected. Also, the characteristic g = 2.08 EPR signal found in some other carbon monoxide dehydrogenases and ascribed to a Ni-Fe-C complex, was never observed by us under any conditions of detection (4 to 100 K) and incubation in the presence of ferricyanide, dithionite, CO, coenzyme A, or acetyl-coenzyme A. Novel, high-spin EPR was found in the oxidized enzyme with effective g-values at g = 14.5, 9.6, 5.5, 4.6, 4.2, 3.8. The lines at g = 14.5 and 5.5 were tentatively ascribed to an S = 9/2 system (approximately 0.3 spins/alpha beta) with rhombicity E/D = 0.047 and D less than 0. The other signals were assigned to an S = 5/2 system (0.1 spins/alpha beta) with E/D = 0.27. Both sets of signals disappear upon reduction with Em,7.5 = - 280 mV. With a very similar reduction potential, Em,7.5 = - 261 mV, an S = 1/2 signal (0.1 spins/alpha beta) appears with the unusual g-tensor 2.005, 1.894, 1.733. Upon further lowering of the potential the putative double cubane signal also appears. At a potential E approximately - 320 mV the double cubane is only reduced by a few percent and this allows the detection of individual cubane EPR not subjected to dipolar interaction; a single spectral component is observed with g-tensor 2.048, 1.943, 1.894.     

Not prostacyclin synthase but prostaglandin endoperoxide synthase increases with human placental development

Prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH sythase) and prostacyclin synthase (PGI synthase were quantitated with specific immunoradimetric assays in microsomes from human placentae (n=20) obtained from 7 up to 17 weeks of gestation. Over that period, wherein trophoblastic invasion of the uterine spiral arteries occurs, the placetae showed a significant increase in concentrations of PGH synthase (r=0.73, p<0.001; n=20), but not in those of PGI synthase. While the variation between individual placentae was much larger for PGI synthase than for PGH synthase concentrations, there was no evidence for a large excess of PGI synthase over that of PGH synthase in any of these early placentae. The data indicate, first, that the developing placenta contains PGI synthase, but in amount which are relatively small and do not appear to increase with advancing gestation. Second, they seem to indicate that the capacity for bioconversion of arachidonic acid into prostaglandin endoperoxides increases markedly with placental development.     

Not prostacyclin synthase but prostaglandin endoperoxide synthase increases with human placental development

Prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantitated with specific immunoradiometric assays in microsomes from human placentae (n = 20) obtained from 7 up to 17 weeks of gestation. Over that period, wherein trophoblastic invasion of the uterine spiral arteries occurs, the placentae showed a significant increase in concentrations of PGH synthase (r = 0.73, p less than 0.001; n = 20), but not in those of PGI synthase. While the variation between individual placentae was much larger for PGI synthase than for PGH synthase concentrations, there was no evidence for a large excess of PGI synthase over that of PGH synthase in any of these early placentae. The data indicate, first, that the developing placenta contains PGI synthase, but in amounts which are relatively small and do not appear to increase with advancing gestation. Second, they seem to indicate that the capacity for bioconversion of arachidonic acid into prostaglandin endoperoxides increases markedly with placental development.     

Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12 degrees C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state. The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]...Tyr+. was suggested as the structure of this two-electron oxidized state of the enzyme. A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C-13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.     

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

Atypical Features of Thermus thermophilus Succinate:Quinone Reductase

The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His₈-SdhB and rcII-SdhB-His₆) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.     

Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins

Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d'Estaintot, Acta Cryst. D50 (1994) 739-743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049-15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV-visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g=4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g=6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g=1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)-Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.     

Trapping a hydrazine reduction intermediate on the nitrogenase active site

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.